This application is in response to Grand Opportunity (GO) RFA-OD-09-004 for Exploratory Research in the Development of Vaccines for AIDS-associated Malignancies. The goal of the application is to develop a novel therapeutic vaccine to Epstein-Barr virus (EBV) and its associated malignancies. EBV infection is responsible for the majority of AIDS-associated lymphomas. We propose to target the viral-encoded protein, EBNA1, which is the only viral protein consistently expressed in all EBV-associated malignancies. The vaccine will incorporate the fusion of the HSV gD protein to EBNA1 to overcome a negative regulatory arm of the adaptive immune response that has been implicated in tumor-associated immune escape. The vaccine vector will be derived from the E1-deleted adenoviral vectors based on the chimpanzee-serotype 68 (AdC68) to eliminate background immunogenicity to more common human serotypes. The vaccine will be tested in rhesus macaques, using the rhesus lymphocryptovirus (rhLCV) as the most appropriate animal model for EBV lymphomagenesis. To complete these goals, we have assembled a team of investigators with expertise in EBV, rhLCV, cancer-vaccine development and analysis, and immunology of non-human primates. For the studies proposed in this GO application, we will focus on a vaccine to rhEBNA1. Most rhesus macaques are naturally infected with rhLCV, and upon their infection with simian immunodeficiency virus (SIV), rhLCV becomes reactivated and can cause lymphomas, suggesting that this virus is an excellent model for proof-of- principle testing of a therapeutic vaccine to EBV. The EBNA1 vaccine will be tested first for the induction of T cell responses in inbred mice, comparing vaccines that express the rhLCV EBNA1 homologue (rhEBNA1) alone or within gD. Once the immunogenicity of the vaccines has been demonstrated in mice, they will be tested in a pilot study in rhesus macaques with pre-existing T cells to EBNA1 due to a natural infection. Immunization success will be measured by the following parameters: a) an increase in cell mediated responses following immunization;b) an increase in antigen specific memory responses to LCV rhEBNA1 in vivo post immunization;c) enhanced quality of the rhEBNA1 directed response based on the frequencies of polyfunctional (multi-cytokine) responses;d) a broader rhEBNA1 T cell repertoire;and e) an increase immune control in virus regression assays and decreased antigen shedding in saliva. These studies will serve as an important proof-of-principle analysis for an EBNA1-specific therapeutic vaccine for treatment of EBV- associated malignancies. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus (EBV) infects over 95% of the adult population worldwide, and has been estimated to be a contributing agent in ~1% of all human cancer. Its carcinogenic potential is significantly elevated by immunosuppression, as is found in the context of HIV-AIDS and tissue transplantation. Evidence suggests that most EBV-associated malignancies arise through defects in the normal cellular mediated immune response. Methods that enhance the EBV-specific cytotoxic T-cell response, like adaptive immunotherapy, can be effective in the treatment of some EBV-associated cancers. Unfortunately, traditional vaccine-strategies have not been successful since these approaches do not overcome the defects in the T-cell response to viral infection. We propose to use a novel strategy to reverse the immunological defect in viral-specific T-cell response using new methods developed in our laboratories. This new vaccine strategy will be tested in mouse and rhesus macaques, which serve as the most relevant immunological models for human infection and viral- carcinogenesis. These pilot studies will provide important information on the mechanism of T-cell response and suppression in EBV-associated cancer, and potentially provide a new vaccine for therapeutic treatment of EBV-associated malignancies in cancer patients.